Sialic acids are derivatives of the nine-carbon sugar neuraminic acid and encompass three parent molecules, N-acetyl-(Neu5Ac), N-glycolyl-(Neu5Gc) and deamino-neuraminic acid (3-deoxy-D-glycero-D-galacto-nonulosonic acid, KDN), which can be substituted at C-4, C-7, C-8 and C-9 by various moieties. They have many major biological roles, ranging from embryogenesis to neural plasticity to pathogen interactions. Although they may rarely occur in free form, they are usually found in chemical covalent linkage at the non-reducing terminus or in internal positions of oligosaccharide side-chains of glycoproteins and glycolipids. The linkages of sialic acids in which they are bound to penultimate sugars such as galactose, N-acetyl-galactosamine and N-acetyl-glucosamine are most commonly α-2,3- and α-2,6-ketosidic bonds.
Among sialoglycoconjugates, sialylated human milk oligosaccharides are of great importance which is directly linked to their unique biological activities such as antibacterial, antiviral, immune system and cognitive development enhancing activities. Sialylated human milk oligosaccharides are found to act as prebiotics in the human intestinal system helping to develop and maintain the intestinal flora. Furthermore they have also proved to be anti-inflammatory, and therefore these compounds are attractive components in the nutritional industry for the production of, for example, infant formulas, infant cereals, clinical infant nutritional products, toddler formulas, or as dietary supplements or health functional food for children, adults, elderly or lactating women, both as synthetically composed and naturally occurring compounds and salts thereof. Likewise, the compounds are also of interest in the medicinal industry for the production of therapeutics. In the human milk oligosaccharides the sialic acid residue is always linked to the terminal 3-O- and/or 6-O-position(s) of D-galactose via α-glycosidic linkage.
The availability of naturally occurring sialylated human milk oligosaccharides is limited. Mature human milk is the natural milk source that contains the highest concentrations of milk oligosaccharides (12-14 g/l), other milk sources are cow's milk (0.01 g/l), goat's milk and milk from other mammals. This low natural availability and difficult isolation methods are important motivations for the development of biotechnological and chemical methodologies for the production of these attractive compounds.
Approximately 200 HMOs have been detected from human milk by means of combination of techniques including microchip liquid chromatography mass spectrometry (HPLC Chip/MS) and matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FT ICR MS) (Ninonuevo et al. J. Agric. Food Chem. 54, 7471 (2006)), from which to date at least 115 oligosaccharides have been structurally determined (Urashima et al.: Milk Oligosaccharides, Nova Medical Books, NY, 2011). These human milk oligosaccharides can be grouped into 13 core units (Table 1). About a quarter of oligosaccharides contains sialic acid.
TABLE 113 different core structures of human milk oligosaccharides (HMOs)NoCore nameCore structure1lactose (Lac)Galβ1-4Glc2lacto-N-tetraose (LNT)Galβ1-3GlcNAcβ1-3Galβ1-4Glc3lacto-N-neotetraoseGalβ1-4GlcNAcβ1-3Galβ1-4Glc(LNnT)4lacto-N-hexaose (LNH)Galβ1-3GlcNAcβ1-3(Galβ1-4GlcNAcβ1-6)Galβ1-4Glc5lacto-N-neohexaoseGalβ1-4GlcNAcβ1-3(Galβ1-4GlcNAcβ1-6)Galβ1-4Glc(LNnH)6para-lacto-N-hexaoseGalβ1-3GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glc(para-LNH)7para-lacto-N-neohexaoseGalβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glc(para-LNnH)8lacto-N-octaose (LNO)Galβ1-3GlcNAcβ1-3(Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-6)Galβ1-4Glc9lacto-N-neooctaoseGalβ1-4GlcNAcβ1-3(Galβ1-3GlcNAcβ1-3Galβ1-(LNnO)4GlcNAcβ1-6)Galβ1-4Glc10Iso-lacto-N-octaoseGalβ1-3GlcNAcβ1-3(Galβ1-3GlcNAcβ1-3Galβ1-(iso-LNO)4GlcNAcβ1-6)Galβ1-4Glc11para-lacto-N-octaoseGalβ1-3GlcNAcβ1-3 Galβ1-4GlcNAcβ1-3Galβ1-(para-LNO)4GlcNAcβ1-3Galβ1-4Glc12Lacto-N-decaoseGalβ1-3GlcNAcβ1-3[Galβ1-4GlcNAcβ1-3(Galβ1-(LND)4GlcNAcβ1-6)Galβ1-4GlcNAcβ1-6]Galβ1-4Glc13Lacto-N-neodecaoseGalβ1-3GlcNAcβ1-3[Galβ1-3GlcNAcβ1-3(Galβ1-(LNnD)4GlcNAcβ1-6)Galβ1-4GlcNAcβ1-6]Galβ1-4Glc
The isolation of sialooligosaccharides form human and other mammals' milk is rather difficult even in milligram quantities due to the presence of a large number of similar oligosaccharides. To date only analytical HPLC methodologies have been developed for the isolation of some sialooligosaccharides from natural source.
The synthesis of complex sialooligosaccharides follows multistep synthetic pathways utilising protection and deprotection strategies. Stereoselective chemical synthetic processes can become very complicated due to the extensive use of protecting groups. These strategies give sialylated oligosaccharides via stereoselective O-sialylation of appropriate protected glycosyl acceptors using glycosylhalide, thioglycoside or diethylphosphite donor activations. The use of either very expensive or very toxic chemicals for the sialylation such as mercury cyanide, mercury bromide and silver carbonate is one of the reasons that make these methodologies less attractive. Inefficient stereocontrol and/or poor yields likewise make(s) the strategies less suitable for further developments. Additionally, these strategies are characterized by severe purification difficulties.
In the case of enzymatic production of sialooligosaccharides, sialyltransferases and sialidases have been the preferred enzymes used. These complex enzymatic systems represent very expensive methodologies for scale-up production and difficult purification protocols are likewise a hindrance for further technology developments. Sialidases could not be used successfully in large scale production methodologies due to low yields and lack of regio- and stereoselectivity. Although in some cases sialyltransferase enzymes are found to be effective in the synthesis of complex sialooligosaccharides (e.g. the synthesis of 1-O-β-benzyl glycoside of 3′-O—(N-acetyl-neuraminosyl)-lactose sodium salt: WO 96/32492; the synthesis of 1-O-β-(4,5-dimethoxy-2-nitro)-benzyl glycoside of 3′-O—(N-acetyl-neuraminosyl)-lactose sodium salt: Cohen et al. J. Org. Chem. 65, 6145 (2000)), the need of CMP-activated sialic acid (cytidine 5′-monophosphosialic acid) as sialyl donor—whose availability is, in fact, rather limited—for transferring the sialic acid portion to the acceptor oligosaccharide restricts their usefulness.
The ability of N-acetyl-lactosamine benzyl glycoside and benzyl glycosides of mucin oligosaccharides from T. cruzi to act as substrate in transsialidase reaction has been studied (Lubineau et al. Carbohydr. Res. 300, 161 (1997); Agusti et al. Bioorg. Med. Chem. 15, 2611 (2007)).
Regioselective sialidation of unprotected or anomerically substituted galactose, lactose or N-acetyl-lactosamine derivatives by means of sialidases in poor yield has been reported (Thiem et al. Angew. Chem. Int. Ed. Eng. 30, 1503 (1991); Schmidt et al. Chem. Comm. 1919 (2000); Schmidt et al. J. Org. Chem. 65, 8518 (2000)).
Some biotechnological methodologies are also described using genetically modified bacteria, yeasts or other microorganisms. Such methods have serious drawbacks in regulatory processes due to limiting commercialisation opportunities.
Sialoglycoconjugates are known to be unstable under certain reaction conditions, such as to acid and base. Indeed, they are able to self-hydrolyse. Accordingly, conditions for preparation and purification of these compounds must be carefully selected.
In summary, isolation technologies have never been able to provide large quantities of sialooligosaccharides due to the large number of oligosaccharides present in the pool of natural origin, e.g. in human milk. Additionally, the presence of regioisomers characterized by extremely similar structures further made separation technologies unsuccessful. Enzymatic methodologies suffer from the low availability of enzymes, extremely high sugar nucleotide donor prices and regulatory difficulties due to the use of enzymes produced in genetically modified organisms. The preparation of oligosaccharides via biotechnology has huge regulatory obstacles due to the potential formation of several unnatural glycosylation products. Generally, all the chemical methods developed for the synthesis of sialooligosaccharides have several drawbacks which prevented the preparation of even multigram quantities of the target compounds (e.g. see the synthesis of 3′-O- and 6′-O-(N-acetyl-neuraminosyl)-lactose through the corresponding benzyl glycoside: Rencurosi et al. Carbohydr. Res. 337, 473 (2002)).
During the past decades the interest in the preparation and commercialisation of sialylated human milk oligosaccharides has been increasing steadily. There is still a need for novel methodologies which can simplify preparation and overcome or avoid purification problems encountered in prior art methods.